Comparison of approaches for microscopic imaging of skin lymphatic vessels

Assessment of skin lymphatic vessels is of great significance in understanding their roles in many pathological conditions. Our aim was to identify the optimal approach for investigation of cutaneous lymphatic system. We performed comparative studies on skin lymphatic vessels using immunohistochemistry of tissue sections, computer graphic reconstruction method together with immunohistochemically stained serial sections and whole mount fluorescence in human lower limb. Lymphatic vessels were identified with podoplanin antibody. The relative merits and drawbacks of each method in evaluation of structure, spatial organization, and distribution of cutaneous lymphatic vessels were described. Immunohistology of tissue sections enabled the investigation of the structure and distribution of the whole cutaneous lymphatic system in two-dimensional slices, whereas three-dimensional morphology of only the most superficial lymph capillary network immediately under the epidermis could be evaluated with the whole mount technique. Meanwhile, only little segmentation of skin lymphatic vessel from five immunohistochemically stained serial sections was reconstructed and evaluated due to expense and special skills required using computer graphic three-dimensional reconstruction. Furthermore, a great number of artifacts and special skills required in its processes leaded to less accurate structure of skin lymphatic vessels. Our findings demonstrated that the use of either of the proposed techniques alone could not allow a comprehensive analysis of the skin lymphatic system due to their relative drawbacks. Combination of immunohistology of tissue sections and three-dimensional whole-mount preparations appears to be the best candidate for comprehensive evaluation of skin lymphatic system.     

The use of silver nitrate as a stain for scanning electron microscopy of arterial intima and paraffin sections of kidney

The suitability of silver nitrate as a stain for scanning electron microscopy was investigated. Accordingly en face preparations of arterial intima were impregnated with silver nitrate, fixed with formalin, and coated with platinum-palladium alloy. In SEM images, the silver lines surrounding the endothelial cells, and the deposits on the intima appear as white lines or dots on a darker background. Similar results were noted for nitrocellulose-embedded endothelium. Paraffin sections of kidney treated with silver nitrate (von Kossa's stain) were also examined. Deposits of the calcium salts of phosphates and carbonates (stained with silver nitrate) were easily differentiated from other tissue components. Results were compared with those obtained with the light microscope. The scanning electron microscopic examination provided a finer definition of the silver granules relative to the surrounding architecture. The limitations and advantages of these techniques and possible further applications of silver nitrate as a stain for scanning electron microscopy are discussed. The use of silver nitrate as a stain for some SEM preparations is recommended.     

Immunogold-silver staining of lymphocyte surface antigens on cells in suspension and in lymph node cryostat sections

An immunogold–silver staining (IGSS) technique for the light microscopical detection of leucocyte cell surface antigens in cell suspensions and cryostat sections is described. The specimens were first incubated with monoclonal mouse antibodies and then with colloidal gold-labelled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained and mounted. In light microscopy, the tissue architecture and the cellular morphology were well preserved. Positive cells showed dark granules on their surface membranes. Optimal labelling conditions were determined. This method proved to be a reliable tool for the enumeration of T-cells and their subsets in peripheral blood. The dense labelling permitted the use of panoptic counterstains like May-Grünwald-Giemsa or Wright's stain. This IGSS technique was used to determine the distribution of the T- and B-cell subsets in cryostat sections of reactive lymph nodes. The sensitivity of the method was comparable with that of immunofluorescence microscopy for cell suspensions and that of the biotin–avidin–peroxidase technique for tissue sections. Immunogold–silver staining was combined with enzyme cytochemistry. In dark-field or epipolarization microscopy the labelling appeared as bright granules on a dark background. With its dense granular membrane labelling and its good morphology IGSS is an ideal method for the study of particular cell types in mixed cell suspensions. In addition, it could be a general method for the detection of cell surface antigens in all kinds of cells and tissues.     

Identification of nerve fibres in the scanning electron microscope.

The use of silver as a "stain" for nerve fibres in scanning electron microscopy (SEM) preparations has been investigated. Samples of cardiac tissues were treated according to the Bielschovsky silver impregnation method. Following embedding in paraffin, successive sections were selected for light microscopy and for SEM studies, respectively. The silver impregnated fibres, when examined in SEM preparations, appeared nearly white against a greyish background of cardiac tissue. They were therefore easy to localize even at a low magnification. These nerve fibres were identified in the same tissue, but different blocks by means of transmission electron microscopy (TEM) studies and by fluorescence microscopy.     

Dynamics of Golgi impregnation in neurons.

This paper describes the early stages of impregnation by the Golgi rapid method in sections and blocks of brain tissue. Aldehyde-fixed and potassium dichromate-treated sections of cerebral cortex were placed on glass slides and coverslipped. The dichromate solution was then replaced by a silver nitrate solution, and events taking place in the section were monitored and time-lapse recorded until the impregnation was interrupted and the sections subsequently prepared for electron microscopy. The tissue blocks, fixed and chromated in the same way, were placed into a silver nitrate solution for 30 min to 24 h and the progress of impregnation compared with the results obtained in the sections on the glass slides. Two basic modes of impregnation were observed, apparently in direct relation to the process of crystallization of silver chromate: crystals of silver chromate growing directly from the surface of the tissue into the nerve cell via its transected plasma membranes, and microcrystalline precipitate of silver chromate spreading into the nerve cell from nucleation centres dispersed in the tissue. The precipitate grows inside the cell as in a preformed channel until the cell has been filled. If the nucleation begins extracellularly, the precipitate extends into the narrow intercellular gaps. Electron microscopy showed that the crystalline precipitate consisted of multilamellar formations containing dense coalesced granules that did not cross plasma or endocellular membrane boundaries.     

Far-field plasmonic resonance enhanced nanoparticle image velocimetry within a microchannel

In this paper, a novel far-field plasmonic resonance enhanced nanoparticle-seeded particle image velocimetry has been demonstrated to measure the velocity profile in a microchannel. Chemically synthesized silver nanoparticles have been used to seed the flow in the microchannel. By using discrete dipole approximation, plasmonic resonance enhanced light scattering has been calculated for spherical silver nanoparticles with diameters ranging from 15 to 200 nm. Optimum scattering wavelength is specified for the nanoparticles in two media: water and air. The diffraction-limited plasmonic resonance enhanced images of silver nanoparticles at different diameters have been recorded and analyzed. By using standard particle image velocimetry techniques, the velocity profile within the microchannel has been determined from the images.     

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523).     

Seventh Report of the Nomenclature Committee

By using standard cine-micrography apparatus in conjunction with a simple turntable under the microscope it is possible to locate and orient serial sections for sequential photography on cine film. The location of the sections is sufficiently accurate to enable the film to be projected at normal speeds. The effect is to produce a steady image giving the viewer the impression of travelling through the specimen. This can assist in the interpretation of serial sections and in the three-dimensional reconstruction of microscopic structures. The film loops thus obtained can also be used directly for teaching purposes, allowing students to obtain quickly the mental images required for a three-dimensional understanding of microstructure. The method can be used in interpreting any series of sections without special preparation. It is particularly valuable for embryological studies and for investigating elongated sub-structures such as blood vessels.     

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification

Light microscopic autoradiographs of H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.     

Lymphatic networks in the periodontal tissue and dental pulp as revealed by histochemical study.

The structural organization and fine distribution of the lymphatic networks in the periodontal tissues (gingiva, periodontal membrane, and alveolar process) and dental pulp of animals and humans were reviewed with special reference to histochemical examination by light and electron microscopy. The distinction between lymphatics and blood vessels was made on cryostat sections of undecalcified and calcified teeth treated with EDTA solution and whole mount preparations of periodontal membranes using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. This staining procedure allowed lymphatic vessels in the periodontal tissue and dental pulp to be differentiated from blood vessels. The specificity and localization of the enzyme reactions were confirmed by comparative histochemical studies of the same specimen with light microscopy and scanning or transmission electron microscopy. Well-developed 5'-Nase-positive lymphatic networks were observed on the tissue sections and whole mount preparations of the gingiva, periodontium, and dental pulp. More lymphatic vessels were seen in the root area of the periodontium than in the cervical area. In the dental pulp, lymphatic vessels were more numerous in the central part than in the peripheral odontoblastic layer. These distributions of the lymphatic capillary networks are discussed in relation to their ability to supply lymph to the teeth.     

Peltate trichomes on biogenic silvery leaves of Elaeagnus umbellata

External and internal features of Elaeagnus umbellata leaves were investigated by optical and electron microscopy. The adaxial (upper) and abaxial (lower) leaf surfaces appeared green and silver in color, respectively. There were peltate trichomes on both the adaxial and abaxial leaf surfaces. The peltate trichomes were 200–300 μm in diameter and shield or umbrella‐shaped. They had a central dome and 20–30 radiating rays that were fused to form a circular cap in the center and tapered at the end. The density of peltate trichomes was apparently higher on the abaxial leaf surface than on the adaxial leaf surface. At least two layers of peltate trichomes were commonly observed on the abaxial leaf surface. The epidermal cells on the abaxial leaf surface had convex lens‐like shape in cross sections. No distinct chloroplasts were found in the cytoplasm of peltate trichomes. These results suggest that the silver coloration on the abaxial leaf surface is mostly due to structural coloration associated with the profuse overlapping peltate trichomes having a circular cap of radially fused rays. The shrub did not show any pigmentary cellular features associated with the silver coloration. With the silvery leaves as a reflective surface for shaded leaves in canopy, E. umbellata is likely to adapt to the harsh non‐native light‐demanding environments.     

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.     

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.     

Granulated metrial gland cells in the murine uterus: localization,kinetics, and the functional role in angiogenesis during pregnancy

Granulated metrial gland (GMG) cells are a major immune cell population in the murine pregnant uterus, and contribute to the maintenance of pregnancy by functioning as uterus-specific natural killer (NK) cells. In order to reveal their kinetics, activation, and functional roles in pregnancy, we conducted quantitative and immunohistochemical analyses in normal and immuno-modulator-treated mice. Under a light microscope, GMG cells were identified by red cytoplasmic granules in periodic-acid-Schiff (PAS)-stained sections. They progressively increased in number and size with the peak at day 12-14 of pregnancy in the decidua and metrial gland. New vessel formation was most prominent around day 8, and the total vascular area reached the peak at day 13. GMG cells were often located near the blood vessels, and expressed vascular endothelial growth factor (VEGF), suggesting their possible inducing role in angiogenesis during the development of decidua/metrial gland. While blood vessels in the non-pregnant uterus were negative for vascular cell adhesion molecule (VCAM)-1, those in the pregnant one were positive. Treatment with neutralizing antibody against VCAM-1, however, did not decrease the number of GMG cells. On the other hand, mitosis of GMG cells was frequently observed. These data suggest that the increment of GMG cells during pregnancy may largely result from local proliferation in the uterus rather than an increased influx of precursor cells. Although we attempted to induce in vivo activation of GMG cells by administration of interleukin-12 (IL-12) or alpha-galactosylceramide, a potent activator for natural killer-T (NK-T) cells, the number of GMG cells did not appreciably increase. The present study has demonstrated that GMG cells locally proliferate in the pregnant uterus, not being related to VCAM-1 expression by the uterine vasculature or systemic activation of NK cells and NK-T cells, and seem to be involved in angiogenesis in the pregnant uterus through VEGF production.     

The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique

The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.     

Area changes in slices of rat brain during preparation for histology or electron microscopy.

Cerebral slices cut from rat brain, either 2-3 mm or 0.27 mm thick, were used to study the effect of embedding and freezing. Paraffin wax sections 6 micrometer thick were mounted and stained with haematoxylin and eosin or Marsland et al.'s (1954) silver stain, and their areas were examined at each step. Embedding in paraffin wax of slices 2-3 mm thick, or in Epon of slices 0.27 mm thick, caused a diminution of their areas by 20-30%. Staining of paraffin wax sections did not alter their areas. Glycerol alone at 15% concentration had no effect on the areas, but at 30% concentration they were diminished by approximately 20%. Diminution of the areas of glycerol treated slices 0.27 mm thick also occurred when they were transferred to liquid N2 or to isopentane, but the areas increased after glycerol was replaced by Freon 12. It was concluded that embedding or freezing cerebral slices caused changes in their areas, but that staining of sections after they had been embedded, sectioned and mounted did not.     

Preservation of ultrastructure and immunoreactivity in cryosections of brain tissue stored in a sucrose-gelatin solution at freezing temperatures

We evaluated the preservation of ultra-structure and immunoreactivity in cryosections of central nervous system tissue mounted with and stored in a sucrose–gelatin solution for one month at −20°C or −80°C. The ultra-structure of synaptic structure in these sections was well preserved and comparable to that of freshly cut cryosections. Quantitative analysis of mitochondrial ultra-structure demonstrated gradually lower degrees of preservation in sections stored at −20°C and −80°C compared with that in freshly cut sections. We observed distinct metabotropic glutamate receptor 1 (mGluR1)-immunogold labelling at peri-synaptic sites in freshly cut sections and also in those stored at −20°C and −80°C. Quantitative analysis of mGluR1 immunoreactivity revealed that the total number of immunogold particles per synapse and the number of non-specifically bound particles were similar under all three conditions. However, the percentage of gold particles bound to a specific synaptic region was greatest in freshly cut sections (79.0%) and progressively lower in sections stored at −20°C (76.1%), in which sections were not frozen, and in sections stored at −80°C (68.0%). These data indicate that ultra-thin cryosections may be conveniently stored in a sucrose–gelatin solution at −20°C for cryoultramicrotomy-immunolabelling.     

Contrast definition and contour detection for logarithmic images

Logarithmic images, such as images obtained by transmitted light or those produced by the human visual system, differ from linear images. Their processing and analysis require consequently specific laws and structures. The latter have been developed in the concept of a logarithmic image processing (LIP) model (Jourlin & Pinoli, 1987, 1988; Pinoli, 1987a). This model permits the introduction of a well-justified contrast definition: from a physical point of view, it is closely linked with logarithmic images and from a mathematical point of view, it is set up in an algebraic structure. The applications presented at the end of this paper concern image preprocessing and segmentation. In particular, in the case of microscopic images, the proposed method of segmentation gives good results with transmitted light (thin foils in biology or transmitted electronic microscopy). However, images obtained by reflected light microscopy are not within the scope of this model.     

Techniques for converting Golgi precipitate in CNS neurons into stable electron microscopic markers.

Direct electron microscopy of nervous tissue stained with the Golgi impregnation method is unsatisfactory because the cytoplasm of the cell bodies and processes of the impregnated neurons are completely filled with a compact precipitate of electron dense silver chromate. This precipitate entirely obscures the cytological details of the impregnated neurons. Because of its solidity and instability in aqueous solutions, the silver chromate is also a source of inconvenience during the preparation of the ultrathin sections. This review summarizes methods that have been developed with the aim of replacing the Golgi precipitate in CNS neurons with a more convenient electron dense material--for example, heavy metal salts or metallic particles. Conversion of the precipitate into a stable electron dense marker is done before the material is embedded for electron microscopy. The methods include lead, gold, and bromide substitution, treatment with ammonia, direct chemical reduction into metallic silver, and photoreduction of the silver chromate into silver through irradiation with ultraviolet light.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.